The long-term objective of the proposed research is to further elucidate the biochemical relationships contributing to the formation of cataracts. The studies proposed for the next three years have been suggested by research already completed or still in progress. A stable isotope method has been developed by which several pathways of amino acid metabolism can be monitored simultaneously in a single lens using gas chromatography-mass spectroscopy (GCMS). In previous experiments with rat lenses cultured with 15N-glutamate, it was possible to demonstrate 15N incorporation into alanine, aspartate, glycine, proline, and serine. This method will be used to test the hypothesis that aging and cataractogenesis will cause measurable changes in lens amino acid metabolism, and that these changes will in turn have an impact upon other closely coupled metabolic pathways. GCMS will be used to determine which of the pathways of amino acid metabolism are important in normal human and animal lenses and to study how these pathways are affected by aging and cataractogenesis. The concentration and 15N enrichment of lenticular free amino acids will be determined following culture of intact human or animal lenses with 15N-labeled precursor, especially 15N-glutamic acid and (15N-amino)-glutamine. The resulting data will be correlated with variables such as age, cataract, or composition of the culture medium. Specific methods will used to study the individual reactions which couple the important pathways of amino acid metabolism to other metabolic pathways. In particular, radioisotopic and chromatographic methods will be used to study the metabolism of radiolabeled serine, ornithine, and bicarbonate and to determine the metabolic products formed when lenses are cultured with these compounds. Thus GCMS will be used to develope a broad overivew of changes in amino acid metabolism in human lenses, and radioisotopic methods will be used to study individual reactions. Successful completion of these proposed studies will provide a more integrated picture of lens metabolism and how it is affected by aging and cataractogenesis.